Chia seeds ameliorate cardiac disease risk factors via alleviating oxidative stress and inflammation in rats fed high-fat diet

Obesity upsurges the risk of developing cardiovascular disease, primarily heart failure and coronary heart disease. Chia seeds have a high concentration of dietary fiber and increased concentrations of anti-inflammatoryand antioxidant compounds. They are used for weight loss plus enhancing blood glucose and lipid profile. The current perspective was commenced to examine the protective influence of chia seeds ingestion on cardiovascular disease risk factors in high-fat diet-fed rats. Forty male albino rats (with an initial body weight of 180–200 g) were used in this study. Rats were randomly and equally divided into 4 groups: Group I was the control group and group II was a control group with chia seeds supplementation. Group III was a high-fat diet group (HFD) that received HFD for 10 weeks and group IV was fed on HFD plus chia seeds for 10 weeks. In all groups Echocardiographic measurements were performed, initial and final BMI, serum glucose, AC/TC ratio, lipid profile, insulin (with a computed HOMA-IR), creatinine phosphokinase-muscle/brain (CPK-MB), CRP, and cardiac troponin I (cTnI) and MAP were estimated. Whole heart weight (WHW) was calculated, and then WHW/body weight (BW) ratio was estimated. Eventually, a histopathological picture of cardiac tissues was performed to assess the changes in the structure of the heart under Haematoxylin and Eosin and Crossmon’s trichrome stain. Ingestion of a high diet for 10 weeks induced a clear elevation in BMI, AC/ TC, insulin resistance, hyperlipidemia, CRP, CPK-MB, and cTnI in all HFD groups. Moreover, there was a significant increase in MAP, left ventricular end diastolic diameter (LVEDD), and left ventricular end systolic diameter (LVESD). Furthermore, histological cardiac examination showed structural alteration of the normal structure of the heart tissue with an increase in collagen deposition. Also, the Bcl-2 expression in the heart muscle was significantly lower, but Bax expression was significantly higher. Chia seeds ingestion combined with HFD noticeably ameliorated the previously-recorded biochemical biomarkers, hemodynamic and echocardiography measures, and histopathological changes. Outcomes of this report reveal that obesity is a hazard factor for cardiovascular disease and chia seeds could be a good candidate for cardiovascular system protection.


Serum glucose level
The level of serum glucose was estimated by using glucose enzymatic (GOD-PAP)-liquizyme Kits (Biotechnology, Egypt), according to 35 .

Measurement of serum insulin level
The level of serum insulin was estimated via using rat insulin enzyme-linked immunosorbent assay kitBioSource Europe S.A.-Rue de l'Industrie, 4-A-1300 Nivelles-Belgium according to 36 .
Measurement of triglycerides (TG) level in serum.The TG level was estimated following rat triglycerides enzyme-linked immunosorbent assay kit, (BioSource Europe S.A.-Rue de l'Industrie, 8-C-1150 NivellesBelgium according to 38 . Measurement of high-density lipoproteins (HDL) in serum.The HDL level was estimatedusing a rat HDLcholesterol enzyme-linked immunosorbent assay kit, (BioSource Europe S.A.-Rue de l'Industrie, 8-A-1340 Nivelles-Belgium) Measurement of low-density lipoproteins (LDL) in serum.The level of LDL was measured depending on the 39 method.The LDL was computed as follows: LDL=TC-HDL-TG\5.
Measurement of serum C-reactive protein (CRP).The CRP was estimated by using rat Immuno-enzymometric assay kits, (MonobindInc Lake Forest, Ca 92630, USA), as early recorded by 41 .
Measurement of serum creatine phosphokinase muscle/brain (CPK-MB):.The CPK-MB level was calculated by using a commercial kit (CPK-MB Kit, Ultra Group, Egypt) following the protocol of 42 .
Measurement of serum cardiac troponin I (cTnI).The cTnIwas estimated by using a commercial kit (Catalog Number SE120134 collected from Sigma-Aldrich Co.) as previously documented by 43 .E.Macro-morphological,Macro-morphometric, and Histopathologicalpictures: Following the collecting of hematological samples, fixation of animals was performed ventrodorsally then by using a sterile scissor, their thoracic cavities were opened asepticallyto revealthe contents.The sternum was dissected to explore the heart and surrounding pericardial fat from the surrounding lungs and the heart was removed for macro-morphological and macro-morphometrical studies.The pericardial fat in all groups was dissected and their mass was weighed using electrical balance, andthen used for quantitative statistical analysis.
Heart weights are obtained for testing the progress of cardiac hypertrophy.Then the whole body weight (WHW) / body weight (BW ratio) was calculated according to 44 .
The cardiac samples were fixed using buffered neutral formalin (10%), then exposed to dehydration, and cleared in Xylene.Soft melted paraffin was used for infiltrating all specimens then soaked in hard paraffin.Paraffin sections were stained using Harris's Hematoxylin and Eosin (H and E) stain as a standard staining method to reveal the histological picture, Crossmon's trichrome stain (for muscle and collagen fibers) 45 .

Tissue preparation for immunohistochemistry and scoring:
The tissue sections were mounted on slides, and the xylene was used for deparaffinization, then, the sections were re-hydrated, and finally rinsed in phosphate buffer saline (PBS).The sections were soaked in 0.3% hydrogen peroxide in aqueous solution.The sections were then washed in 10% normal rabbit serum to lessen the binding of immunoglobulins 45 .The sections were incubated with antisera containing the primary antibody: secondary antibody (1:500).The slides were incubated at room temperature for 30 minutes then were washed two times with P.B.S, 5 minutes each.The sections were kept in a humidified chamber then, incubated at horse radish peroxidase polymer (1:60 diluted by deionized water) for 30 min at room temperature.Diaminobenzidine (DAB) was utilized as chromogen and sections were exposed to incubation (2-4 min) at room temperature.Sections were cleaned, then counterstained with Mayer's hematoxylin, dehydrated, cleared, and then mounted in Canada balsam 46 .All the photomicrographs were taken by using by a high-resolution camera (Canon Powershot G10 14.7 MP Digital Camera with Digital Photo Professional software) attached via a vertical tube to a standard light microscope (Olympus BX 21) at the Department of Histology and Cytology, Zagazig University.

Scoring
Cardiomyocyte hypertrophy and adipocyte scoring was quantified based on cell count per area (by using H and E stain).While collagen level scoring (by using Crossmon'strichrome stain in order to elevate the accuracy of collagen deposition scoring), and immunohistochemical scoring for the expression of Bax and Bcl2 (by using immune-stained sections) were measured based on staining percentage per area.The scoring system was assessed by examining six slides per each group from the various four groups at 100x magnification then the average scores were calculated.Such system assessed each parameter independently and recorded a score of 0-3.Each sample was taken with a histological score using Abramov's histological scoring system (Table 1) 47 .
Determination of Bcl-2 and Bax mRNA according to 48 Bcl-2 and Bax mRNA Gene Expression Detected by Reverse Transcriptase Polymerase Chain Reaction The myocardium tissues were rinsed in liquid nitrogen, and immersed and stored in -80°C refrigerator for RNA extraction.Total RNA was isolated from frozen myocardium tissue usingTRNzol RNA Reagent Kit (TIANGEN, Beijing, China) according to manufacture protocol.Purified RNA sample were stored at -80°C.The purity and integrity of total RNA were monitored by spectrophotometer, and electrophoresis was carried out on a denaturing formaldehyde agarose gel and stained with ethidium bromide.300ng mRNA was reversely transcripted to single-stranded cDNA using Invitrogen M-MLV cDNA kit according to manufacture protocol.
The 2 × Taq PCR Green Mix kit and specific primers for Bcl-2 , Bax and b-actin (control) were utilized for the PCR reaction assayThe initial denaturation step was at 95°C followed by 33 cycles for bcl-2 and 29 cycles for bax for 30 s, annealing for 30 s at 55°C for bcl-2 and 60°C bax, and extension for 30°C seconds at 37°C.The PCR products were electrophoresed on a 2% agarose gel.The photographed gel was analyzed using the gel document system (gel pro version 0.3)software Media Cybermetica USA.The results were expressed as a ratio of target mRNA/b-actin mRNA an external control for each sample.

Statistical analysis
The expression of the gathered data in the current perspectives represented as mean ± SD for quantitative variables and analyzed statistically via using the Statistical Package for the Social Sciences (SPSS) program (version 24) (SPSS Inc.Chicago, IL, USA.One-way ANOVA was used for comparing the mean values of more than two groups.The test was counted as significant at p-values <0.05.The resulting smaller p-value indicated a higher significant value. For histological scoring, Kruskal-Wallis Test was conducted to examine the differences in cardiomyocyte hypertrophy scoring, adipocytes scoring, collagen level scoring, and immunohistochemical scoring for the expression of Bax and Bcl2 according to the types of groups or treatments taken.Mann Whitney test was performed as a pairwise comparison following a significant Kruskal-Wallis test.P < 0.05 is considered a statistical difference.Statistical analysis was performed by SPSS version 25 (Armonk, NY: IBM Corp) and Graph Pad Prism 8.0.2 (GraphPad Software, Inc).

Ethical approval
All the experimental procedures were conducted in accordance with the guiding principles for the care and use of research animals and were approved by the Institutional Research Board of Faculty of Medicine, Zagazig University (ZU-IACUC/3/F/249/2022).All animal experiments were complied with the ARRIVE guidelines and

Macro-morphological results and anthropometric measures
In the gross anatomical images of hearts, in groups I and group II , amount of pericardial fat surrounding heart are normal (Fig. 1a).Groups (III, IV) accumulated more pericardial fat relative to control rats (groups I, II) (Fig. 1b).However, the amount of fats decreased with chia ingestion (group IV) (Fig. 1c).The HFD increased pericardial fat in group III when compared to groups I and II.While consumption of chia seeds in group IV significantly reversed that effect when compared to group III (Fig. 1d) To assess the development of obesity, we calculated BMI and AC/TC ratio, groups III and IV showed a significant elevation in AC/TC ratio and BMI (p < 0.001) compared to groups I and II.However, these ratios were significantly (p < 0.001) lower in group IV compared to group III (Fig. 2a-c).
There was a clear elevation in heart weight in group III in comparison to groups I, II, and IV (p<0.001).Additionally, the ratio between heart and body weights (H/ BW %) demonstrated no significant difference among groups I, II, III, and IV(0.48±0.01,0.48±0.0,050±0.01,and 0.49±0.09respectively.

Serum biochemical analysis
As an indication of insulin resistance, the fasting blood glucose and insulin concentrations and HOMA were measured which were noticeably elevated in groups III and VI than in groups I and II (p < 0.001).However; blood glucose and HOMA showed lower levels in group IV (p < 0.05)related to group III (Fig. 3a-c).
Regarding biochemical parameters supporting the development of metabolic syndrome, serum TC, LDL,TG, and VLDL levels were estimated and they reported significantly increased values in group III than the group I and group II (p < 0.001), but in group IV the recorded indices were significantly lessened (p < 0.001) when compared with group III.Meanwhile, we reported a significant (P<0.001)reduction in serum HDL in group III compared to groups I and II (Figs. 4, 5a-c).There was a significant increase (p < 0.001) in Atherogenic indices in group III and group IV compared to group I and group II.Group IV exhibited a significant (p < 0.001) decrease in the same indices compared to group III (Fig. 4c).www.nature.com/scientificreports/Regarding tissue inflammation as a result of obesity, There was a significant elevation (P<0.001) in serum CRP in group III and group VI compared to group I and group II, chia seeds ingestion significantly (P<0.01)reduced these levels in group IV compared to the group III (Fig. 6a, b, c).
As an indication of cardiac damage, there was a significant (p < 0.001) increase in CK-MB and cTnI in group III and group VI in comparison with group I and group II.Chia ingestion significantly (P<0.001)reduced these levels in group IV compared to group III (Fig. 6a-c).

Hemodynamic and echocardiography measures
As a metabolic syndrome associated with hypertension, MAP was evaluated and it was recorded a significant (p < 0.001) elevation in group III compared to group I and group II, and these levels were significantly (p < 0.01) reduced in group IV.
For assessmentof the effect of obesity on diastolic functions, the present study clarified that LVESD & LVEDD were significantly augmented in group III (p<0.001) when compared to group I and group II.On the other side, these indices were noticeablyimproved by chia ingestion in group IV (Fig. 7a-c).

Histopathological examination of the heart tissues
The examination of left ventricle sections using a Light microscope revealed these findings: in group I, a highly organized myocardium structure where cardiac muscle fibers appeared cylindrical in shape and extensively branched.They were shorter and usually contained only one nucleus, which was located in the central region of the cell and acidophilic cytoplasm (Fig. 8A).
In group II which fed on a chia diet, the same phenotype was observed and myocardial architecture was normal where cardiac muscle fibers had acidophilic cytoplasm and centrally located nucleus with little collagen fiber between the cardiac muscle bundles (Fig. 8B).www.nature.com/scientificreports/Ingroup III which received a high-fat diet, the high-fat diet induced structural disorganization, accompanied by cardiomyocyte hypertrophy distinguished by a widening in cell surface area, and slightly degenerated cardiac myocytes with paler cytoplasm.There were slightly congested blood capillaries between muscle fibers (Fig. 8C).
In group IV (given a high-fat diet with chia seeds supplementation), the cardiac muscle showed normal histological structure with branched cardiac muscle bundles, acidophilic cytoplasm, single nuclei, and little or no thickening of the matrix around the cardiomyocytesnoticed (Fig. 8D).
By staining the different groups by crossmon's trichrome stain it revealed that group I (Fig. 9A) and group II (Fig. 9B) exhibited minute amounts of collagen fibers around the level of blood vessels.However, in group III, crossmon's trichrome staining displayed interstitial myocardial deposition of collagen fibers and distinct perivascular fibrosis (Fig. 9C).In group IV (Fig. 9D), the collagen fibers were markedly reduced in quantity.
In group III, immunohistochemical analysis exhibited that Bcl-2 expression in heart muscle was noticeably lower, but Bax expression was notably elevated.In group IV which was fed on HFD with chia seeds supplementation, Bcl-2 expression was elevated and Bax expression was decreased markedly in the protected group in comparison to groups I and III (Figs. 10 and 11).

Comparison of the histological scoring of the above-mentioned parameters among the different groups by Kruskal-Wallis Test P < 0.05:
Kruskal-Wallis Test results showed that group III revealed a significant difference compared with groups I, II, and IV in cardiomyocyte hypertrophy, adipocyte count, collagen level, immunohistochemical expression of Bax scoring, and immunohistochemical expression of Bcl2 scoring.Meanwhile, there was a non-significant difference between groups I and II.Group IV showed a non-significant difference compared to group I in all parameters except the immunohistochemical expression of Bax scoring which showed a highly significance difference.In addition, group IV exhibited a non-significant difference compared to group II in all parameters (Table 2, Fig. 12).

Findings of Bcl-2 and Bax mRNA
HFD had been accompanied by clearly reduced myocardial anti-apoptotic Bcl-2 expression, nd increased proapoptotic Bax-expression in comparison to group I (P < 0.001).Additionally, the myocardial Bax/Bcl-2 ratio www.nature.com/scientificreports/ was significantly higher (P < 0.001) in group III compared with group I.However, chia seed ingestion (group IV) revealed a significant (P < 0.001) modulation in these results (Fig. 13).

Discussion
Even though the etiology of obesity varies with environmental, genetic, and lifestyle causes, it is greatly linked to co-morbidities like diabetes, cardiovascular diseases, cancer, hypertension, and sleep disorders [49][50][51][52] .The link between heart disease and obesity is multifaceted including direct and indirect mechanisms controlled by obesityassociated co-morbidities 53,54 .Chia seeds have an augmented concentration of dietary fibers; hence, it has been utilized for the loss of weight and improves lipid picture and blood glucose 55 .They are rich in proteins, fatty acids, minerals, and antioxidants that are healthy for heart 56 .Therefore, the goal of the present report was to assess the cardiovascular protective influence of chia seed supplementation in high-fat diet-induced obesity.
To assess obesity development and its risk factors, researchers use animal models for diet-induced obesity, since these models imitate human obesity compared to genetic models 57 .
BMI is used for estimating the obesity extension; meanwhile, it reveals no information on the distribution of fat, which is highly essential in cardiovascular risk 58 .Therefore, other clinical assessments (e.g., abdominal circumference) have been used with the goal of identifying central obesity 4 .According to our present findings, we report a clear elevation in BMI and AC/TC ratio in HF-supplemented diet.Also, ingestion of a high-fat diet resulted in the following consequences including elevated levels of fasting serum glucose, increased HOMA-IR index, in addition to elevated fasting levels of serum TG, LDL, TC, VLDL, cardiac risk ratio, atherogenic coefficient, and the atherogenic index, plus a clear reduction in HDL levels.It is opined that rats consuming a high-fat diet are more vulnerable to diabetes and accordingly have insulin resistance.Previous studies verified our findings and recorded that the increased fasting blood glucose level and higher HOMA-IR index, strongly suggest the occurrence of metabolic syndrome 59,60 .Moreover, 61 mentioned that feeding an enriched diet in high-fat for www.nature.com/scientificreports/ a period of 10 weeks augmented the level of plasma glucose.In the current study, the elevation of TG could be attributed to increased storage of energy as documented by 62 .Also, the elevated level of cholesterol and the reduction in the level of HDL cholesterol resulted in elevated serum atherogenic coefficient as confirmed by 40 .
In line with an earlier study 63 , reported that a high-fat diet produced higher levels of cholesterol, triglycerides, LDL, and VLDL.The main attention is devoted to the ingestion of chia seeds (15%) in group IV which resulted in a clear reduction in BMI and AC/TC ratios and a clear reduction in fasting serum levels of TC, glucose, TG, VLDL, LDL, and HOMA-IR, Cardiac risk ratio, atherogenic coefficient and atherogenic index besides an increase in HDL levels.Such ameliorations could be dominated by the richness of chia seeds in antioxidants and essential fatty acids that could modulate the alterations induced by consuming a high-fat diet.These outcomes were supported by 15,56,64 who reported that chia seeds are a rich source of dietary fiber that promotes weight loss, improves lipid profile and blood glucose, and reduces blood pressure 15,56,64 .These seeds are rich in 34-36% fibers, which is considered higher compared to other cereals or dried fruit 65,66 , and fatty acids, alpha-linolenic acid (ALA),with a higher ratio 67 , which are useful for controlling metabolic diseases 68 .
In concordance with a previous study 69 , revealed noticeable weight loss by involving chia seeds in bread with a decrease of 500 Kcal daily from the normal diet 70 documented a significant body weight loss in a group of people eating a hypocaloric diet of a reduction (25% Kcal)from the daily energy need and he attributed this reduction in body weight to chia seed intervention.However 71 , revealed the significance of chia seeds in controlling weight gain.Also 72 revealed that chia could enhance the reduction of waist circumference.Concurrently 73 , evaluated glycemic responses of chia seeds via lowering blood glucose concentration in healthy individuals.Moreover, other studies were conducted on Wistar rats that received chia seeds and oil and demonstrated a potential impact of lessening TG and elevating HDL 74,75 .Likewise 18,76 , reported that uptake of chia seed in humans induced a significant decline in TG and LDL, while levels of HDL augmented without any side impacts.
Despite the significant reduction in metabolic parameters in group IV, its insulin level, and atherogenic index were still higher than those of control groups, this can be explained by the supplementation of chia seeds was not accompanied by modification of the ingested diet or life style all through the study duration, moreover, these data might be attributed to the dose of chia seeds ingested or the study duration Ingestion of HFD in the present report induced a noticeable increase in MAP.However, chia intake treatment modulated this alteration.The development of obesity-induced hypertension could be returned to over-activation of the sympathetic nervous system producing hypertension in obese humans and animal models as reported by [77][78][79] assumed the mechanism of action of chia seeds which may involve the presence of peptides acting as angiotensin-converting enzyme inhibitors, and thus improve the blood pressure as reported in type 2 diabetes patients.The bioactive peptides with low molecular weight generated from the hydrolysis of chia seed proteins can suppress the activity of ACE 80 .
Previous studies addressed the beneficial components in chia seeds that exert their potential effect on the physiological status and on cardiac health.They are rich in high amounts of insoluble fiber which has been reported to enhance BP more than soluble fiber 74,81 .The components of chia seeds are helpful in cardiovascular disease by lessening blood pressure, cholesterol, aggregation of platelet, and oxidation 25 .Chia is rich in a mixture of essential fatty acids, in a high omega-3 to omega-6 ratio 20,82 , which is associated with lower BP and cardiovascular disease risk 83,84 , and the mineral content for a 100 g carries 335 mg, 407 mg, and 631 mg of Mg, K, and Ca respectively 46 .Both K 85 and Mg 86 are opined as strong inhibitors of contraction of vascular smooth muscle and as vasodilators that can lessen BP; Ca is verified to enhance lower BP in hypertensive patients 87,88 .
Echocardiography examination to evaluate cardiac function rats showed a clear increase in LVESD and LVESD in comparison to group I. Our outcomes were in line with previous observations that found the longterm receiving of HFD caused cardiac fibrosis and dysfunction of LV in rodents 89,90 .
Obesity is distinguished by low-grade chronic inflammation with over-expression of pro-inflammatory adipokines in adipose tissue 91,92 .The increased circulatory levels of these inflammatory mediators have been associated with hepatocyte stimulation to produce CRP 92 .In addition, elevated levels of CRP are predictive in some cardiac diseases like unstable angina and myocardial infarction 93,94 .Also, high CRP levels are associated with increased coronary heart disease and overall mortality as reported by 95 .
Similarly 96 , revealed that Wistar rats that received high carbohydrate and high-fat diets had a CRP amount doubled in comparison to the rats that received the same diet and enriched in 5% chia seeds.Further works are www.nature.com/scientificreports/recommended to elucidate the mechanism of action of chia seeds with considering inflammatory status.On the other side, these data could indicate an anti-inflammatory condition related to the elevated alpha-linolenic acid (ALA) contents detected in chia seeds 96 .
Our results demonstrated that ingestion of HFD caused significantly elevated CK-MB as well as increased troponin-T levels.However, chia seeds intake significantly ameliorated these levels.These findings were supported by 97 who mentioned that the release of such markers indicated impaired membrane permeability and cell death in many tissues of the body including the heart.Few studies showed the link between obesity and the occurrence of myocardial damage, including coronary artery diseases.Another study supported our findings and suggested that obesity is in positive association with subclinical myocardial injury, following the National Health and Nutrition Examination Survey (NHASNES) III 98 .
The negative effect of HFD on the heart could be returned to the occurrence of several mechanisms including oxidative damage and inflammation generated by mitochondria dysfunction, cardiac steatosis, altered adipokinespicture, and hyperglycemia.All are the major events that can cause cardiac failure as reported by 99,100 .However, the inflammatory cytokines derived from adipose tissue and those generated locally from the circulating macrophages and monocytes, in response to heart and oxidative damage, promote heart inflammation.Consequently, ROS can damage the cardiac cells and produce hypertrophy, inflammation, and apoptosis by stimulating various damaging pathways (i.e.NF-κB,MAPKs, TGF-β1/Smad2/3, JNK,ASK1, and ANGII) 101,102 .
Previous reports recorded that measures of obesity, such as body mass index, could be accompanied by higher cTn levels in the general public 103,104 .Another study reported significantly elevated serum levels of Troponin-1 and CK-MB in rats that received HFD 105 .
On the tissue level, the hearts in rats fed a standard control diet showed normal histological structure and were well vascularized.Similar results were documented by 106 report.Also, we reported the occurrence of structural disorganization, slight degeneration, and paler cytoplasm in the supplemented group in high-fat.Similarly 107 , reported that a high-fat diet caused collagen deposits in spaces between cells, cardiomyocytes disorganization, adipose tissue deposits under the pericardium, around vessels, and between cardiomyocytes, interstitial edema, associated with the accumulation of infiltrating cells suggesting cardiomyopathy.The observed cardiac hypertrophy could be induced as a primary step in the sequence of adaptive responses of the cardiac tissue to stress induced by a high number of pathological and physiological conditionsas reported by 108 .Supplementation in chia seeds regenerated the tissue and markedly enhanced these alterations.The same findings were recorded by 106 in rats.Based on the findings of Bcl-2 expression, the current study revealed significantly lower expression in heart muscle in the HFD group, meanwhile, Bax expression was significantly higher, suggesting activation of apoptotic processes.It is suggested that myocardial apoptosis can commence by ingestion of a high-fat diet and accumulation of fatty acid metabolites, myocardial, and activation of inflammatory processes, and cardiac insufficiency 109 .Also, in line with another report, Bax can antagonizeBcl-2 function and control the Bcl-2 ability for prolonging the survival of cells as mentioned by 110 .On the other side, dietary intervention in chia seeds produced significant Bcl-2 expression reflecting the potential antioxidant and anti-inflammatory properties.
Apoptosis is seldom in normal myocardium; meanwhile, it can be activated in both chronic and acute heart pathological conditions 111 .Apoptotic signaling produces apoptosis, via three complex pathways including the mitochondrial-mediated pathway, which included Bcl-2 associated X (Bax) protein, B-cell leukemia/lymphoma-2 (Bcl-2) family, and the Bcl-2,which is opined to be essential in controlling apoptosis 112,113 .The ability of antiapoptotic Bcl-2 to suppress the activity of proapoptotic Bax was dependent on the ratio of Bax to Bcl-2 and on the expression of protein levels 48 .The ratio of Bax to Bcl-2 expression represents a cell death switch, which determines the survival or death of cells in response to an apoptotic stimulus; an increased in this ratio decreases in the cellular resistance to apoptotic stimuli, leading to increased death of cells 114 .Here in, high fat diet increased the pro-apoptotic Bax-to-Bcl-2 gene ratio suggesting occurrence of apoptosis in groups that received high-fat diets, however, the dietary uptake of chia seeds (15%) attenuate this alteration where it elevates the cell resistance via its antioxidant components.
Outcomes of this report reveal that HFD-induced obesity is a risk factor for cardiovascular disease and chia seeds (15% of diet weight) could be a good candidate for cardiovascular system protection.

Figure 1 .
Figure 1.A photo macrograph of rat showing normal amount of pericardial fat surrounding heart representing groups I and II (a), high amount of pericardial fat surrounding heart representing high fat diet as group III (b) , enhancement and decreases amount of pericardial fat representing high fat diet treated with chia as group IV (c) and and Whiskers plot graph shows pericardial fat mass (gm) in all groups, a= vs group I; b= vs group II; c= vs group III; *=significant (P<0.001) and #= significant (P<0.01)(n=10) (d).

Figure 8 . 4 Figure 9 .
Figure 8. Photomicrograph of the heart (A) Group I showing a highly organized myocardium "arrow" with blood vessel "winged arrow".(B) Group II showing normal myocardial architecture "arrow" with blood vessel "winged arrow".(C) group III displaying structural disorganization, cardiomyocyte hypertrophy "zigzag arrows", slightly degenerated of cardiac myocytes with paler cytoplasm "curved arrow" and pale cross sectional cardiac muscle fibers "arrow head", also showing slightly congested vessels "arrow" (D) In group IV, the cardiac muscle showed normal histological structure with branching cardiac muscle bundles "arrow" with blood vessel "winged arrow".Stain: H and E.

Figure 10 .Figure 11 .
Figure 10.Photomicrograph of the heart immunostainedagainst Bax antibody (A) group I showing little reaction "arrow" (B) group II showing mild reaction "arrow" (C) group III showing significantly higher reaction "arrows" (D) group IV showing mild reaction "arrow".

Figure 12 .
Figure 12. (A) Showing a comparison of the cardiomyocyte hypertrophy scoring among the different groups of rats.A minimum of 6 slides were assessed for each group.(B) Showing a comparison of the adipocytes count scoring among the different groups of rats.A minimum of 6 slides were assessed for each group.(C) Showinga comparison of the collagen level scoring among the different groups of rats.A minimum of 6 slides were assessed for each group.(D) Showing a comparison of the immunohistochemical expression of Bax scoring among the different groups of rats.A minimum of 6 slides were assessed for each group.(E) showing a comparison of the immunohistochemical expression of Bcl2 scoring among the different groups of rats.A minimum of 6 slides were assessed for each group.a= vs group I; b= vs group II; c= vs group III; *=significant difference (P<0.05);$= highly significant difference (P<0.01).

Table 1 .
Showing histological score based on Abramov's histological scoring system.
were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments.

Table 2 .
Showing results of histological scoring.